Alexandrium tamarense is a unicellular dinoflagellate protist that causes harmful algal blooms (HABs) and paralytic shellfish poisoning through the production of saxitoxins. Very little is known about the factors that influence the formation of HABs, their recent spread to new areas, and the genes involved in toxin production. Our work also shows that dinoflagellates are outstanding models for understanding endosymbiosis (organelle genesis) and horizontal gene transfer because of their unparalleled ability to host different plastid endosymbionts and to accumulate genes through horizontal gene transfer. Here we propose to generate a significant genomics resource for Alexandrium to accelerate research on the biology and control of HABs and on nuclear genome evolution in dinoflagellates. Our specific aims are to use a highly efficient gene discovery strategy to generate a comprehensive expressed sequence tag (EST) unigene set for Alexandrium. Phylogenomic and detailed phylogenetic analysis of the different sequences in the Alexandrium unigene set will result in trees that help elucidate the contribution of horizontal gene transfer from endosymbionts and other sources to dinoflagellate nuclear genome evolution. We will also use massively parallel signature sequencing analysis to create a transcriptome tag database for Alexandrium cells grown in different culture conditions that affect nutrient physiology, cell and life cycle stages, and toxin production. This work will help us identify target genes involved in these functions and will form the basis for future microarray analyses. This proposal is an integrated, collaborative effort that relies on expertise in molecular evolution, EST processing, and phylogenomics (Bhattacharya, Scares), high quality cDNA library construction (Scares), and expertise in the ecology and physiology of Alexandrium (Anderson). The results of this study will result in a valuable molecular resource for scientists working to understand the ecology and toxicity of HAB species like Alexandrium and will provide the first comprehensive genomics resource for these fascinating protists. [unreadable] [unreadable] [unreadable]